Neurofilaments (NF) enable axons to achieve the large calibers required for normal nerve impulse conduction and play other critical roles in neuronal function only now being appreciated. In several major neurodegenerative diseases, including a form of fronto-temporal dementia, abnormal accumulations of NF are a neuropathologic hallmark believed to contribute to neuronal dysfunction. Indeed, the association of NF gene mutations with Charcot Marie Tooth type 2E/1F neuronopathy and with early-onset Parkinson's disease (PD) unequivocally implicates NF directly in neurodegenerative disease pathogenesis. Our studies in the past grant term revealed new fundamental properties of NF and suggested a novel function for NF as a scaffold in axons and synapses, which reversibly docks vesicular organelles and proteins, including neurotransmitter receptors, and thereby may regulate their function. We identified the middle subunit of NF (NFM) as uniquely crucial for NF transport and key NF scaffold functions. We propose to test hypotheses, based on preliminary data, that the head and rod domains of NFM regulate NF transport and trigger NF network formation along axons. By replacing endogenous NFM in neurons with genetically modified and truncated forms of NFM, we propose to define roles of the head and rod domains, including head phosphorylation, and the effects on NF behavior of a NFM mutation associated with severe PD (aims 1a-d). Studies will be performed in primary neuronal cultures using videomicroscopy and in retinal ganglion cells in vivo by established radiolabeling and ultrastructural methods, exploiting the previously successful approach we used to define functions of NF carboxyl- tail domains. Having pinpointed NFM rod as the likely binding site for NF motors, we will perform genetic screens to identify putative NF motor(s) (aim 1e). Based on new findings that NFM and the D1 dopamine receptor (D1R) interact functionally to modulate D1R -mediated behaviors in vivo, we will test the hypothesis that D1R docking to a NFM-containing scaffold influences endocytic recycling of D1R to synaptic membranes and thus sensitivity to DA agonists (aim 2). We will characterize D1R recycling in striatal primary neurons of mice with NFM deletion or related alterations and will ultrastructurally and biochemically localize D1R-NFM interaction sites within neurons in vivo. Finally, we will clarify the poorly understood role of proteolysis in regulating NF accumulation and NF network stability and its modulation by NF structural properties as a basis for understanding pathological NF accumulation in neurological diseases and brain aging (aim 3). Novel approaches to alter the in vivo activities of three major proteolytic systems implicated by our preliminary data and protease modulations in primary neurons will be used. These studies address fundamental aspects of NF biology related to the normal functions of axons and synapses and are directly relevant to mechanisms of neuroaxonal degeneration in neurofibrillary diseases, including Alzheimer's disease and related dementias, amyotrophic lateral sclerosis and related neuronopathies, Parkinson's disease and glaucoma. PUBLIC HEALTH RELEVANCE: This project addresses how neurons assemble an internal supporting framework enabling them to connect and communicate with other neurons. The regulation of this process controls important neuron functions and is known to be disrupted in neurodegenerative diseases associated with aging. Defining the specific steps in this regulation will help to clarify mechanisms leading to major neurological diseases, including dementias, and will identify novel cellular targets for developing innovative therapies for these diseases.